We recently discovered Kos1 (Kinase of the embryonic stem cells), a novel, nonreceptor protein tyrosine kinase (NRPTK) in differentiating murine embryonic stem (ES) cells following withdrawal of LIF. Kosl is a ubiquitously expressed 47kDa protein, whose forced expression in either mouse or human cell lines, including NIH 3T3, T47D and NCI-H82 cells, is associated with inhibition of growth and apoptosis. Kos1 was shown to autophosphorylate and its catalytic activity is required for its growth inhibitory function. Thus, expression of a kinase null version of Kos1 [Kos1 (K148A)CN] promotes NIH 3T3 cell growth even in liquid and soft agar cultures, indicating Kosl may function as a negative regulator of growth. Preliminary studies also indicate that Kos1 but not Kos1 (K148A)CN can indirectly inhibit Ras and the downstream Raf-MAPK growth signal pathway. Therefore, we hypothesize that Kos1 functions as a negative regulator of cell growth in a mechanism involving inhibition of Ras and may have tumor suppressor properties. Thus, Kos1 may play a role in cancer. To test this hypothesis two specific aims have been identified: 1) To determine how Kos1 is regulated and how it regulates Ras activity and cell growth. How is Kos1 catalytically activated and what are the required Kos1 tyrosine-phosphorylation site(s)? Is tyrosine phosphorylation of Kos1 required for its growth and Ras inhibitory functions? 2) To determine whether Kos1 is required for growth suppression and whether Kos1 can affect growth and development in vivo. Is Kos1 a tumor suppressor? State of the art molecular and biochemical methodologies will be employed to facilitate the studies outlined including mutational analysis of Kos1, in vivo and in vitro expression studies of Kos1 mutants, interactive cloning strategies using Kos1 as "bait" to identify binding/regulatory proteins, and the creation of a Kos1 null mouse through targeted homologous recombination to assess its role in negative growth regulation in vivo. Results are expected to fill in fundamental gaps in our knowledge regarding how NRPTKs and Kos1 specifically may negatively regulate the Ras-Rafl-MAPK growth pathway and thereby potentially aid in the future development of novel anti-cancer therapeutic strategies targeting Kos1.